Chiara Lico

Lutein is needed for efficient chlorophyll triplet quenching in the major LHCII antenna complex of higher plants and effective photoprotection in vivo under strong light.

BackgroundLutein is the most abundant xanthophyll in the photosynthetic apparatus of higher plants. It binds to site L1 of all Lhc proteins, whose occupancy is indispensable for protein folding and quenching chlorophyll triplets. Thus, the lack of a visible phenotype in mutants lacking lutein has been surprising.ResultsWe have re-assessed the lut2.1 phenotypes through biochemical and spectroscopic methods. Lhc proteins from the lut2.1 mutant compensate the lack of lutein by binding violaxanthin in sites L1 and L2. This substitution reduces the capacity for regulatory mechanisms such as NPQ, reduces antenna size, induces the compensatory synthesis of Antheraxanthin + Zeaxanthin, and prevents the trimerization of LHCII complexes. In vitro reconstitution shows that the lack of lutein per se is sufficient to prevent trimerization. lut2.1 showed a reduced capacity for state I – state II transitions, a selective degradation of Lhcb1 and 2, and a higher level of photodamage in high light and/or low temperature, suggesting that violaxanthin cannot fully restore chlorophyll triplet quenching. In vitro photobleaching experiments and time-resolved spectroscopy of carotenoid triplet formation confirmed this hypothesis. The npq1lut2.1 double mutant, lacking both zeaxanthin and lutein, is highly susceptible to light stress.ConclusionLutein has the specific property of quenching harmful 3Chl* by binding at site L1 of the major LHCII complex and of other Lhc proteins of plants, thus preventing ROS formation. Substitution of lutein by violaxanthin decreases the efficiency of 3Chl* quenching and causes higher ROS yield. The phenotype of lut2.1 mutant in low light is weak only because rescuing mechanisms of photoprotection, namely zeaxanthin synthesis, compensate for the ROS production. We conclude that zeaxanthin is effective in photoprotection of plants lacking lutein due to the multiple effects of zeaxanthin in photoprotection, including ROS scavenging and direct quenching of Chl fluorescence by binding to the L2 allosteric site of Lhc proteins.

Plant-produced potato virus X chimeric particles displaying an influenza virus-derived peptide activate specific CD8+ T cells in mice

Plant viruses can be genetically modified to produce chimeric virus particles (CVPs) carrying heterologous peptides. The efficacy of plant-produced CVPs in inducing antibody responses specific to the displayed peptide has been extensively demonstrated. To determine if plants can be used to produce CVPs able to activate peptide-specific major histocompatibility complex (MHC) class I-restricted CD8+ T cells, potato virus X (PVX) has been engineered to display the H-2D(b)-restricted epitope ASNENMETM of influenza A virus nucleoprotein (NP). Engineering criteria were devised to comply not only with plant virus genetic stability and infectivity but also with antigen processing rules. The immunological properties of different doses of endotoxin-free preparations of CVPs or unmodified PVX have been evaluated by s.c. immunizing C57BL/6J mice and testing at different time intervals splenocyte responses by interferon gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay. These experiments demonstrated that CVPs activate ASNENMTEM-specific CD8+ T cells. Remarkably, the best response was achieved without adjuvant co-delivery. These results represent the proof of concept that well-designed plant virus carriers of epitopes produced in plant can reasonably be used into peptide vaccine formulations aimed to activate cell-mediated immune responses.

In planta production of two peptides of the Classical Swine Fever Virus (CSFV) E2 glycoprotein fused to the coat protein of potato virus X

BackgroundClassical Swine Fever (CSFV) is one of the most important viral infectious diseases affecting wild boars and domestic pigs. The etiological agent of the disease is the CSF virus, a single stranded RNA virus belonging to the family Flaviviridae.All preventive measures in domestic pigs have been focused in interrupting the chain of infection and in avoiding the spread of CSFV within wild boars as well as interrupting transmission from wild boars to domestic pigs. The use of plant based vaccine against CSFV would be advantageous as plant organs can be distributed without the need of particular treatments such as refrigeration and therefore large areas, populated by wild animals, could be easily covered.ResultsWe report the in planta production of peptides of the classical swine fever (CSF) E2 glycoprotein fused to the coat protein of potato virus X. RT-PCR studies demonstrated that the peptide encoding sequences are correctly retained in the PVX construct after three sequential passage in Nicotiana benthamiana plants. Sequence analysis of RT-PCR products confirmed that the epitope coding sequences are replicated with high fidelity during PVX infection. Partially purified virions were able to induce an immune response in rabbits.ConclusionPrevious reports have demonstrated that E2 synthetic peptides can efficiently induce an immunoprotective response in immunogenized animals. In this work we have showed that E2 peptides can be expressed in planta by using a modified PVX vector. These results are particularly promising for designing strategies for disease containment in areas inhabited by wild boars.

Potato virus X movement in Nicotiana benthamiana: new details revealed by chimeric coat protein variants

Potato virus X coat protein is necessary for both cell-to-cell and phloem transfer, but it has not been clarified definitively whether it is needed in both movement phases solely as a component of the assembled particles or also of differently structured ribonucleoprotein complexes. To clarify this issue, we studied the infection progression of a mutant carrying an N-terminal deletion of the coat protein, which was used to construct chimeric virus particles displaying peptides selectively affecting phloem transfer or cell-to-cell movement. Nicotiana benthamiana plants inoculated with expression vectors encoding the wild-type, mutant and chimeric viral genomes were examined by microscopy techniques. These experiments showed that coat protein-peptide fusions promoting cell-to-cell transfer only were not competent for virion assembly, whereas long-distance movement was possible only for coat proteins compatible with virus particle formation. Moreover, the ability of the assembled PVX to enter and persist into developing xylem elements was revealed here for the first time.

The Two-Faced Potato Virus X: From Plant Pathogen to Smart Nanoparticle.

Potato virus X (PVX) is a single-stranded RNA plant virus, historically investigated in light of the detrimental effects on potato, the world’s fourth most important food commodity. The study of the interactions with cells, and more generally with the plant, both locally and systemically, significantly contributed to unveil the mechanisms underlying gene silencing, fundamental not only in plant virology but also in the study of gene expression regulation. Unraveling the molecular events of PVX infection paved the way for the development of different viral expression vectors and consequential applications in functional genomics and in the biosynthesis of heterologous proteins in plants. Apart from that, the ease of manipulation and the knowledge of the virus structure (particle dimensions, shape and physicochemical features) are inspiring novel applications, mainly focused on nanobiotechnology. This review will lead the reader in this area, spanning from fundamental to applied research, embracing fields from plant pathology to vaccine and drug-targeted delivery, imaging and material sciences. Due to the versatile moods, PVX holds promise to become an interesting nanomaterial, in view to create the widest possible arsenal of new “bio-inspired” devices to face evolving issues in biomedicine and beyond.

In vitro and in vivo toxicity evaluation of plant virus nanocarriers

The use of biological self-assembling materials, plant virus nanoparticles in particular, appears very intriguing as it allows a great choice of symmetries and dimensions, easy chemical and biological engineering of both surface and/or internal cavity as well as safe and rapid production in plants. In this perspective, we present an initial evaluation of the safety profile of two structurally different plant viruses produced in Nicotiana benthamiana L. plants: the filamentous Potato virus X and the icosahedral Tomato bushy stunt virus. In vitro haemolysis assay was used to test the cytotoxic effects, which could arise by pVNPs interaction with cellular membranes, while early embryo assay was used to evaluate toxicity and teratogenicity in vivo. Data indicates that these structurally robust particles, still able to infect plants after incubation in serum up to 24h, have neither toxic nor teratogenic effects in vitro and in vivo. This work represents the first safety-focused characterization of pVNPs in view of their possible use as drug delivery carriers.

A plant derived multifunctional tool for nanobiotechnology based on Tomato bushy stunt virus

Structure, size, physicochemical properties and production strategies make many plant viruses ideal protein based nanoscaffolds, nanocontainers and nano-building blocks expected to deliver a multitude of applications in different fields such as biomedicine, pharmaceutical chemistry, separation science, catalytic chemistry, crop pest control and biomaterials science. Functionalization of viral nanoparticles through modification by design of their external and internal surfaces is essential to fully exploit the potentiality of these objects. In the present paper we describe the development of a plant derived multifunctional tool for nanobiotechnology based on Tomato bushy stunt virus. We demonstrate the ability of this system to remarkably sustain genetic modifications and in vitro chemical derivatizations of its outer surface, which resulted in the successful display of large chimeric peptides fusions and small chemical molecules, respectively. Moreover, we have defined physicochemical conditions for viral swelling and reversible viral pore gating that we have successfully employed for foreign molecules loading and retention in the inner cavity of this plant virus nanoparticles system. Finally, a production and purification strategy from Nicotiana benthamiana plants has been addressed and optimized.

Plant-Derived Chimeric Virus Particles for the Diagnosis of Primary Sjögren Syndrome

Plants are ideal for the production of protein-based nanomaterials because they synthesize and assemble complex multimeric proteins that cannot be expressed efficiently using other platforms. Plant viruses can be thought of as self-replicating proteinaceous nanomaterials generally stable and easily produced in high titers. We used Potato virus X (PVX), chimeric virus particles, and Cowpea mosaic virus, empty virus-like particles to display a linear peptide (lipo) derived from human lipocalin, which is immunodominant in Sjögren’s syndrome (SjS) and is thus recognized by autoantibodies in SjS patient serum. These virus-derived nanoparticles were thus used to develop a diagnostic assay for SjS based on a direct enzyme linked immunosorbent assay format. We found that PVX-lipo formulations were more sensitive than the chemically synthesized immunodominant peptide and equally specific when used to distinguish between healthy individuals and SjS patients. Our novel assay therefore allows the diagnosis of SjS using a simple, low-invasive serum test, contrasting with the invasive labial biopsy required for current tests. Our results demonstrate that nanomaterials based on plant viruses can be used as diagnostic reagents for SjS, and could also be developed for the diagnosis of other diseases.

Molecular farming for antigen (vaccine) production in plants

Genomic and proteomic approaches to the study of fundamental cell mechanisms are rapidly contributing to broaden our knowledge on metabolic pathways for the optimal exploitation of the cell as a factory. In the last few years this knowledge has led to important advances in the large scale production of diagnostic and therapeutic proteins in heterologous hosts (bacteria, yeasts, mammalian and insect cells or transgenic animals and plants), allowing the comparison of the most efficient methods in terms of costs, product quality and safety.

A biodistribution study of two differently shaped plant virus nanoparticles reveals new peculiar traits.

Self-assembling plant virus nanoparticles (pVNPs) have started to be explored as nanometre-sized objects for biomedical applications, such as vaccine or drug delivery and imaging. Plant VNPs may be ideal tools in terms of biocompatibility and biodegradability endowed with a wide diversity of symmetries and dimensions, easy chemical/biological engineering, and rapid production in plants. Recently, we defined that icosahedral Tomato bushy stunt virus (TBSV) and filamentous Potato virus X (PVX) are neither toxic nor teratogenic. We report here the results of an interdisciplinary study aimed to define for the first time the biodistribution of unlabelled, unpegylated, underivatized TBSV and PVX by proved detecting antibodies. These data add new insights on the in vivo behaviour of these nano-objects and demonstrate that the pVNPs under scrutiny are each intrinsically endowed with peculiar properties foreshadowing different applications in molecular medicine.